On-spot selectively activated hydrophobic slide and preparation thereof

ABSTRACT

An on-spot selectively activated hydrophobic slide/microarray. The preparation method relates to a hydrophobic copolymer prepared by blending, grafting or co-polymerization of a hydrophobic material and a compound bearing a functional group protected by a protecting group, wherein the functional group is imide or cyclic amide, and the protecting group is a photo acid group such as a tosyloxy group. The hydrophobic copolymer coated on a substrate is then subjected to selective photolithographical activation so that the slide will have functional active copolymer spots separated by inactive copolymers. The resulting slide is suitable for the preparation of high-density and high-efficiency bio-chip/microarray.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of the continuation-in-partapplication Ser. No. 10/115,103 which is a continuation-in-part of U.S.patent application Ser. No. 09/695,254 filed on Oct. 25, 2000, whichhereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an on-spot selectively activatedhydrophobic slide/microarray and the preparation method thereof. Moreparticularly, it relates to a hydrophobic copolymer prepared byblending, grafting or co-polymerization of a hydrophobic material and acompound bearing functional groups protected by a protecting group. Thehydrophobic copolymer is coated on a substrate and subjected toselective photolithographical activation to form an on-spot selectivelyactivated hydrophobic slide. The slide can then be used to produce anon-spot selectively activated hydrophobic microarray.

2. Description of the Related Arts

In current biochip and bio-microarray technology, most preparationmethods involve the treatment of a matrix surface with silanization,followed by crosslinking reaction with biomaterials. In the silanizationtreatment, the surface of the substrate is activated based on itsmaterial, and then treated by a hydrophilic silane such as APTES(amino-propyl-tetraethoxy-siliane). Afterwards, the crosslinkingreaction is performed via a crosslinker such as glutaraldehyde toimmobilize biomaterials on the substrate. The shortcomings of thismethod include substrate dependence, long reaction time, poorhomogeneity, low reaction efficiency, and the resulting low activity forthe immobilized biomaterials. Moreover, the prepared covalent bondingsurface is hydrophilic, which facilitates crossover and contaminationamong spots when the hydrophilic surface is used for a high-densitymicroarray.

U.S. Pat. No. 5,837,860 and WO 98/39481 disclose the treatment of glassor silicon wafer with hydrophobic silane such as mercapto-silane, andthe immobilization of nucleic acid probes thereon. The method involvestreating a substrate surface so that mercapto-groups (HS—) withhydrophobicity are covalently bonded thereon. The hydrophobic propertyis suitable for the immobilization of nucleic acids/nucleotides in highdensity. The method, however, requires the modification of thebiomaterials to bear mercapto-groups, thereby forming disulfide bondsbetween the modified biomaterials and matrix surface. Blanchar, A. P. etal. (Biosensors and Bioelectronics, 1996, 11(6/7): 687-690) disclosescoating photoresists onto a substrate and then development using themicro-electromechanical mask to form on-spot hydrophilic spots, whereinthe region outside of the spots is hydrophobic. The preparation ofhigh-density nucleic acid probe microarrays and in situ synthesis iscarried out on this treated surface.

In this prior art, blending, grafting or co-polymerization of ahydrophobic material and a compound bearing functional groups such asimide, cyclic amide, and to prepare a hydrophobic copolymer is notdisclosed. Further, the application of the prepared hydrophobiccopolymer onto an organic or inorganic substrate to form an on-spothydrophilic enhanced slide is also not disclosed.

SUMMARY OF THE INVENTION

It is therefore a primary object of the present invention to provide anon-spot selectively activated hydrophobic slide that allows highlyefficient manufacturing of high-density microarray. The slide of thepresent invention comprises a substrate on which a hydrophobic copolymerlayer is coated. The hydrophobic copolymer layer comprises functionalactive copolymer spots separated by inactive copolymer. The functionalactive copolymer comprises a hydrophobic material and a compound bearinga functional group which is imide or cyclic amide. The inactivecopolymer comprises a hydrophobic material and a compound bearing afunctional group protected by a protecting group, wherein the functionalgroup is imide or cyclic amide, and the protecting group is a photo acidgroup. Photo acids are acid groups that are sensitive to light ofspecific wave length and will convert into other compounds when exposeto the light. Examples of photo acid groups include, sulfonyloxy group,N-methanesulfonyloxy group, N-trifluoro-methanesulfonyloxy group,camphorsulfonyloxy group and tosyloxy group.

Another aspect of the present invention provides an on-spot selectivelyactivated hydrophobic microarray comprising a substrate coated with ahydrophobic copolymer layer having functional active copolymer spotsseparated by inactive copolymers, and a biologically active materialimmobilized on the functional active copolymer spots. The functionalactive copolymer comprises a hydrophobic material and a compound bearinga functional group which is imide or cyclic amide. The inactivecopolymer comprises a hydrophobic material and a compound bearing afunctional group protected by a protecting group, wherein the functionalgroup is imide or cyclic amide, and the protecting group is a photo acidgroup.

Yet another aspect of the present invention provides a method forpreparing an on-spot selectively activated hydrophobic slide with thefollowing steps. First, a hydrophobic copolymer is prepared in a solventto obtain a solution of hydrophobic copolymer. The solution ofhydrophobic copolymer is then coated on a substrate (e.g. an organic orinorganic substrate). The solvent is then removed. The hydrophobiccopolymer layer on the substrate is subjected to selectivephotolithographical activation to form a hydrophobic copolymer layerhaving functional active copolymer spots separated by inactivecopolymers.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more fully understood and furtheradvantages will become apparent when reference is made to the followingdescription of the invention and the accompanying drawings in which:

FIG. 1 is a diagram showing the fluorescent intensity afterimmobilization of labeled oligonucleotide probe using a glass slidecoated with poly(styrene-co-[N-(tosyloxy)maleimide]) withoutphotolithographical activation as a matrix.

FIG. 2 is a diagram showing the fluorescent intensity afterimmobilization of labeled oligonucleotide probe using a glass slidecoated with poly(styrene-co-[N-(tosyloxy)maleimide]) withphotolithographical activation as a matrix.

FIG. 3 is a diagram showing the fluorescent intensity of three differentoligonucleotide probes hybridized with their targets respectively,wherein the oligonucleotide probes are immobilized on a glass slidecoated with poly(styrene-co-[N-(tosyloxy)maleimide]) and treated byphotolithographical activation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features a method for preparing an on-spotselectively activated hydrophobic slide with the following steps. First,a hydrophobic copolymer is prepared in a solvent to obtain a solution ofhydrophobic copolymer. The solution of hydrophobic copolymer is thencoated on a substrate (e.g. organic or inorganic substrate). The solventis then removed. The hydrophobic copolymer layer on the substrate isthen subjected to selective photolithographical activation to form ahydrophobic copolymer layer having functional active copolymer spotsseparated by inactive copolymer (ie., the hydrophobic copolymer).

In accordance with the method of the present invention, the hydrophobiccopolymer is prepared by blending, grafting or co-polymerization of ahydrophobic material and a compound bearing a functional group protectedby a protecting group. Examples of the functional group are imide orcyclic amide. The protecting group is a photo acid group such assulfonyloxy group, N-methanesulfonyloxy group,N-trifluoro-methanesulfonyloxy group, camphorsulfonyloxy group andtosyloxy group, preferrable is tosyloxy group. The hydrophobic copolymersolution is then coated onto an organic or inorganic substrate followedby removing the solvent to form a hydrophobic layer with covalentbonding functional groups protected by the protecting groups. Theresulting hydrophobic layer is comparatively inert due to the existenceof the large protecting group.

During the photolithographical activation step, the hydrophobic layer iscovered with a photo mask that selectively exposes predetermined areas(or spots) of the hydrophobic layer. These exposed areas of thehydrophobic copolymer layer are subject to photolithographicalactivation with light at an appropriate wavelength to remove theprotecting groups from the functional groups of the hydrophobiccopolymers. The resulting functional active copolymers have functionalgroups capable of forming covalent bond with biological materials. Thehydrophobic copolymers covered by the photo mask maintain the protectedform and are inert to react with biological materials. Therefore, when amicroarray is prepared using such slide, the biological material willonly bind to the functional active copolymer spots but not to theinactive areas between the spots.

A main feature of the present invention is that the functional activecopolymer spots are separated by inactive copolymers. With theapplication of the photo mask and photolithographical activation, thedistance between the spots can be reduced without mixing materials to beimmobilized on different spots. The density of spots of a resultingmicroarray is therefore largely increased.

In addition, the covalent bonding reaction between the functional activecopolymer and the biological material is reduced to one-step reaction sothat the immobilization efficiency is improved. Further, the compoundsbearing functional groups (e.g. imide or cyclic amide) or thederivatives thereof protected with the protecting groups can be blended,grafted, or co-polymerized with hydrophobic material in various ratiosto adjust the density of functional groups on the resulting functionalhydrophobic copolymer spots.

Another feature of the present invention is that when the structure ofimide or cyclic amide is attacked by a biomaterial or a modifiednucleophile, another hydrophilic group will be formed resulting in theformation of on-spot hydrophilic enhancement. It is therefore beneficialto enhance not only the covalent bonding on the microarray but also thespecificity of subsequent biochemical reaction, thereby improving thechoke point of the traditional hydrophilic silanization.

The term “on-spot hydrophilic enhanced” used herein refers to theformation of another hydrophilic group via ring-opening when thestructure of imide or cyclic amide on the hydrophobic matrices preparedby the present invention is attacked by a nucleophile (e.g. aminemodified oligonucleotide probe), which leads to the formation of on-spothydrophilic enhancement. At this point, the hydrophobic surface isconverted to hydrophilic, whereas the other region remains hydrophobic.

When the protected hydrophobic copolymer ispoly(styrene-co-[N-(tosyloxy)maleimide]) and the functional hydrophobiccopolymer is PSMI, the aforementioned reactions are shown as Scheme I:

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In addition, the abbreviationsthroughout the specification have the following meanings: APTES,amino-propyl-tetraethoxy-siliane; PS, polystyrene; MI, maleimide; PSMI,poly(styrene-co-maleimide); PE, polyethylene; PEMI,poly(ethylene-co-maleimide); Ts, tosyloxy group; TsCl, toluenesulfonicacid chloride; TsOMI, N-(tosyloxy)maleimide; IPA, isopropyl alcohol; andAIBN, N,N′-azobisisobutyronitrile. The chemical formula of Ts group isshown as following:

According to the method for preparing an on-spot selectively activatedhydrophobic slide of the present invention, the type of the substrateused herein is not limited, and can include an organic or inorganicsubstrate (i.e. substrate-independent). Organic substrates include apolymer polymerized by organic monomers. Suitable organic monomersinclude, for example, ethylene, styrene, propylene, ester, acrylic acid,acrylate, alkyl acrylic acid, or alkyl acrylate. Inorganic substratesinclude, but are not limited to, silicon wafer, ceramic material, glassor metal. The substrate can further comprises a polymer consisting ofpoly(styrene-co-maleimide), or poly(ethylene-co-maleimide) if desired.Also, the substrate can further comprises a photoactive polymer having aphoto acid group if desired.

In one preferred embodiment, the hydrophobic copolymer prepared by thepresent invention (as described below) can be directly molded byinjection or compression to form an on-spot hydrophilic enhanced slidewith hydrophobic surface, wherein the technique of injection orcompression molding is well known to those skilled in this art. Suitablehydrophobic copolymers used herein include, but are not limited to,poly(styrene-co-maleimide).

If an inorganic substrate is employed, an activation of the substratesurface can be carried out prior to coating hydrophobic copolymersthereon to enhance the adhesion between the substrate surface andhydrophobic copolymers. The activation step includes treatment of thesubstrate surface with an acid or a base, or treatment of the surface byplasma activation.

According to the preparation method of the invention, the substratesurface can be cleaned prior to coating hydrophobic copolymers thereonto prevent the deposition of impurities or contaminants on the substratesurface. The cleaning step is performed by pretreatment with a solventand/or sonication, based on the material of the substrate. Suitablesolvents include, but are not limited to, surfactant, water, alcohol oracetone.

In accordance with the present invention, the hydrophobic copolymer isprepared by blending, grafting or co-polymerization of a hydrophobicmaterial and a compound bearing a functional group protected by aprotecting group, wherein the functional group is imide or cyclic amide,and the protecting group is a photo acid group such as sulfonyloxygroup, N-methanesulfonyloxy group, N-trifluoro-methanesulfonyloxy group,camphorsulfonyloxy group and tosyloxy group, preferrable is tosyloxygroup. The hydrophobic material can include any compound that blends,grafts or co-polymerizes with an imide, such as maleimide,4-amino-phthalimide; or cyclic amide, such as 3-methyl-2-pyridone. Suchhydrophobic materials include styrene, urethane, ethylene, orderivatives thereof. In a particularly preferred embodiment of thepresent invention, the hydrophobic material used is polystyrene orethylene, and the prepared hydrophobic copolymers thus include PSMI, orPEMI.

The method for coating the hydrophobic copolymer onto a matrix is notlimited, and is understood by one of ordinary skill in the art to whichchemical engineering and semiconductor process belongs, to include spincoating, screen printing, roller coating, curtain coating, or dipcoating, etc. In one preferred embodiment, the coating method usedherein is spin coating, preferably at 3,000-6,000 rpm, and morepreferably at 4,000 rpm.

After coating the hydrophobic copolymer onto a substrate, the excesssolvent is removed by means of, for example, vacuum evaporation, heatingevaporation, or evaporation under reduced pressure, wherein the methodof heating evaporation is carried out at a temperature not higher than100° C. to prevent the matrix from being destroyed or to preventundesired polymerization. The preparation of the slide of the inventionis accompanied after the solvent is removed.

The hydrophobic copolymer coated on the substrate is then subjected toselective photolithographical activation. In the present invention, theprotecting group is a photo acid such as sulfonyloxy group,N-methanesulfonyloxy group, N-trifluoro-methanesulfonyloxy group,camphorsulfonyloxy group and tosyloxy group, preferrable is tosyloxygroup, and the protected functional groups of the hydrophobic copolymeris very inert to biological materials. In the present invention, thepreferred wavelength of the light used for photolithographicalactivation is 248 nm or 312 nm. The functional group of the hydrophobiccopolymer is activated by appropriate photolithography, which removesthe protecting group from the functional group of the hydrophobiccopolymer and thus activate the functional group. The functional activegroup is capable of forming covalent bond(s) with a biological material.

In a preferred embodiment of the present invention, a photo mask is usedduring the photolithographical activation process to define the areas(spots) to be activated. When different biological materials are to beimmobilized on one slide to form a microarray, different photo masks canbe used. That is, for the first biological material, predetermined spotson the hydrophobic copolymer layer are activated using a first photomask followed by immobilization of the first biological material, andthen a second photo mask is used to activate spots on which the secondbiological material is immobilized, and so on. An advantage of thisselective photolithographically activated slide is that the activatedand thus functional active copolymer spots are separated by inactive(ie., protected) hydrophobic copolymers, and therefore greatly increasesthe density of the resulting microarray. The employment of photo mask(s)also facilitates the manufacture of high density microarray.

In another aspect of the present invention, an on-spot selectivelyactivated hydrophobic microarray is provided. The microarray comprises abiologically active material, which is immobilized on the on-spotselectively activated hydrophobic slide described above. Theimmobilization is achieved by way of contacting the bio-molecules withthe on-spot selectively activated hydrophobic of the present invention.The biologically active material has a nucleophile, such as an aminegroup, present in the molecule (e.g. a protein) itself or in achemically-modified entity thereof. The linkage between the nucleophileof the biologically active material and functional groups of thefunctional active copolymer is formed via the ring-opening reaction.

Bio-molecules used as the biologically active material that are suitablefor use in the invention include nucleic acid, oligonucleotide, peptidenucleic acid (PNA), antigen, antibody, enzyme, or protein. Stable amidelinkages are formed after such bio-molecules are reacted with thefunctional groups of the on-spot hydrophilic enhanced slide of theinvention. As compared with the prior arts in which the bonding iscreated via a two-step reaction (i.e., by the silane-based polymer andfollowed by adding a crosslinker such as glutaldehyde), the reaction isreduced to one-step reaction in the present invention. Thereby, the timefor the immobilization reaction is substantially decreased andefficiency is increased.

Another feature of the present invention is the property of on-spothydrophobic/hydrophilic dynamic conversion when the slide is applied toimmobilization of the biomaterials. In addition, a high-densitymicroarray can be prepared according to the matrix with a functionallayer of hydrophobic characteristic, and the biomaterials' orientationduring subsequent reaction can be improved by way of the on-spothydrophilic conversion. Further, the preparation time according to themethod of the invention is much less than that of the conventionalmethod, and the homogeneity of the slide of the present invention isincreased. In other words, the number of the functional groups (e.g.imide or cyclic amide) appearing on the matrix and the bonding strengthis average. Moreover, the on-spot hydrophilic enhanced microarray of thepresent invention can markedly decrease the time required forimmobilization of biomaterials onto the matrix. In one preferredembodiment, the immobilization time is less than 40 minutes.

Without intending to limit it in any manner, the present invention willbe further illustrated by the following examples.

EXAMPLE 1 Preparation of TsOMI

0.25 g N-hydroxymaleimide and 0.5 g TsCl are mixed in 50 ml toluene/IPAsolution. The reaction mixture was heated to 60° C. and allowed to reactin the presence of N₂ for 3 hours to give TsOMI solution.

EXAMPLE 2 Preparation of poly(styrene-co-[N-(tosyloxy)maleimide])

The procedure for preparing poly(styrene-co-[N-(tosyloxy)maleimide]) isshown in Scheme II: Scheme II:

To a pre-filled nitrogen or argon reaction bottle containing toluene,polystyrene and TsOMI were added in a molar ratio of 4:1, followed byaddition of 0.5% N,N′-azobisisobutyronitrile (AIBN) dropwise. Theco-polymerization was conducted at 60° C. for 2 hours, and then ceasedby aeration to obtain poly(styrene-co-[N-(tosyloxy)maleimide]) intoluene.

EXAMPLE 3 Effectiveness of Oligonucleotide Immobilization ofpoly(styrene-co-[N-(tosyloxy)maleimide])

Poly(styrene-co-[N-(tosyloxy)maleimide]) synthesized from Example 2 wasdissolved in toluene and then coated on glass slides at 4,000 rpm toform poly(styrene-co-[N-(tosyloxy)maleimide]) slides. The slides weredried in an oven at 100° C. to remove toluene. A syntheticoligonucleotide probe in which the 5′ end was labeled with fluorescenceand the 3′ end bore amine group, was immobilized to the aforementionedslides to perform a ring-opening reaction. The immobilization conditionswere described as follows: 0.5 μM of the probe in 2×SSC buffer (pH 7.0)was spotted on the slides and incubated at 37° C. for 1 hour. The slideswere washed with 0.2% SDS for 10 minutes. The fluorescence analyses wereperformed for the slides with and without washing (control) to monitorthe immobilization efficiency. The result is shown in FIG. 1. It isshown that the amount of oligonucleotide probes immobilized onpoly(styrene-co-[N-(tosyloxy)maleimide]) is very low.

EXAMPLE 4 Effectiveness of Oligonucleotide Immobilization ofpoly(styrene-co-maleimide)

Poly(styrene-co-[N-(tosyloxy)maleimide]) slides were prepared asdescribed in Example 3. After the toluene dried, the slides were exposedto a light of 248 nm wavelength for 10 minutes to convert poly(styrene-co-[N-(tosyloxy) maleimide]) to PSMI. Next, a syntheticoligonucleotide probe was immobilized on the slides as described inExample 3 to perform a ring-opening reaction. The fluorescence analyseswere performed for the slides with and without washing (control) tomonitor the immobilization efficiency. The result is shown in FIG. 2. Itis shown that the amount of oligonucleotide probes immobilized onpoly(styrene-co-maleimide) is much higher compared with the results fromExample 3.

EXAMPLE 5 Hybridization with Specific Oligonucleotides

The slides used in this Example were the same as those described inExample 4. The oligonucleotides used in this example were AlO₃ (composedof 29 nucleotides with 15 Thymidines and amine group at the 5′ end); O₃(composed of 29 nucleotides without amine group at the 5′ end); and P₃(composed of 29 nucleotides with amine group at the 5′ end),respectively. The probe used for labeling hybridization reaction was thecomplementary sequence thereto, wherein the 3′ end was labeled withfluorescence. The immobilization conditions were 2×SSC, pH 7.0; 3×SSC,pH 7.0; and 10×SSC, pH 3.0, respectively, and the immobilization timewas 1 hour. The hybridization reaction was performed for 4 hours. Thefluorescence was analyzed to monitor the hybridization result. Theresult is shown in FIG. 3.

The functional active copolymers of the present invention, namely PSMI,and PEMI, persist reaction functionalities (e.g. imide ring) to whichthe biomaterials can be bonded, and therefore a bio-molecule such asamine-modified DNA can be stably bonded to the surface of the slides. InFIG. 1, since poly(styrene-co-[N-(tosyloxy)maleimide]) has itsfunctional site(s) protected by a large photo acid group, tosyloxy, mostof the oligonucleotide probes did not form stable bonding with thecopolymer and were washed out after washing. As compared with thephotolithographically activated slides on whichpoly(styrene-co-[N-(tosyloxy)maleimide]) has been converted to PSMI(FIG. 2), the oligonucleotide probe retains at least 90% immobilizationefficiency after washing with 0.2% SDS.

In traditional methods, the immobilization of oligonucleotide probeoften requires 4-16 hours. However, by the method of the presentinvention, only about 30 minutes is required to achieve the same effectof probe immobilization. This gives the present invention anothersignificant advantage over the prior techniques.

In FIG. 3, three different probes were tested for their effectiveness ona photolithographically activated slide. The AlO₃ probe is composed of29 nucleotides with 15 Thymidines and amine group at the 5′ end, the O₃probe is composed of 29 nucleotides without amine group at the 5′ end,and the P₃ probe composed of 29 nucleotides with amine group at the 5′end. These probes were immobilized on a photolithographically activatedslide at pH 3.0 and 7.0, respectively. Poor bonding efficiency of O₃probe was observed due to the lack of an amine group at its 5′ end.Other probes with amine group at the 5′ end possessed excellent bondingefficiency. The result indicates that bonding specificity of a targetmolecule can be elevated markedly through the presence of a 5′ end aminegroup at the corresponding probe.

The density of functional groups of the functional active copolymerspots is adjustable by adjusting the proportions of hydrophobic materialand protected functional compounds during the synthesis of thehydrophobic copolymer. When the density of a functional group increases,the immobilization efficiency is also increased. On the other hand, thecopolymer becomes more hydrophilic, which is not desirable in thepreparation of high density microarray. Therefore, the density offunctional groups in the hydrophobic copolymer layer should be adjustedaccording to different conditions.

Homogeneity is also an important fact in the preparation of microarrays.The hydrophobic copolymer of the present invention has been shown tohave excellent homogeneity after coating to the substrate.

The component of the hydrophobic copolymer is not limited tostyrene/polystyrene. The copolymer with polyethylene (PE) as the mainchain can also be developed into an on-spot hydrophilic enhanced slide.Any functionally hydrophobic copolymer containing at least one imide orcyclic amide can attain the purpose of the present invention.

While the invention has been particularly shown and described with thereference to the preferred embodiment thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade without departing from the spirit and scope of the invention.

1. An on-spot selectively activated hydrophobic slide, comprising: a substrate; and a hydrophobic layer coated on the substrate, the hydrophobic layer comprising functional active copolymer spots separated by inactive copolymer, wherein the functional active copolymer comprises a hydrophobic material and a compound bearing a functional group which is imide or cyclic amide, and the inactive copolymer comprises a hydrophobic material and a compound bearing a functional group protected by a protecting group, wherein the functional group is imide or cyclic amide, and the protecting group is a photo acid group.
 2. The slide as claimed in claim 1, wherein the photo acid group is a sulfonyloxy group, N-methanesulfonyloxy group, N-trifluoro-methanesulfony-loxy group, camphorsulfonyloxy group or tosyloxy group.
 3. The slide as claimed in claim 1, wherein the photo acid group is a tosyloxy group.
 4. The slide as claimed in claim 1, wherein the hydrophobic material is styrene, urethane, ethylene, or derivatives thereof.
 5. The slide as claimed in claim 1, wherein the functional active copolymer comprises poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 6. The slide as claimed in claim 1, wherein the inactive copolymer comprises poly(styrene-co-[N-(tosyloxy)-maleimide]), or poly(ethylene-co-[N-(tosyloxy)-maleimide]).
 7. The slide as claimed in claim 1, wherein the substrate comprises a polymer polymerized by organic monomers, wherein the organic monomers are ethylene, styrene, propylene, ester, acrylic acid, acrylate, alkyl acrylic acid, or alkyl acrylate.
 8. The slide as claimed in claim 1, wherein the substrate comprises a polymer consisting of poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 9. The slide as claimed in claim 1, wherein the substrate comprises an inorganic material of silicon wafer, ceramic material, glass, or metal.
 10. The slide as claimed in claim 1, wherein the substrate further comprises a photoactive polymer having a photo acid group.
 11. An on-spot selectively activated hydrophobic microarray, comprising: a substrate; a hydrophobic layer coated on the substrate, the hydrophobic layer comprising functional active copolymer spots separated by inactive copolymer, wherein the functional active copolymer comprises a hydrophobic material and a compound bearing a functional group which is imide or cyclic amide, and the inactive copolymer comprises a hydrophobic material and a compound bearing a functional group protected by a protecting group, wherein the functional group is imide or cyclic amide, and the protecting group is a photo acid group; and a biologically active material comprising a nucleophile immobilized to the functional active copolymer via a ring-opening reaction between the functional group of the hydrophobic functional copolymer and the nucleophile of the biologically active material.
 12. The microarray as claimed in claim 11, wherein the photo acid group is a sulfonyloxy group, N-methanesulfonyloxy group, N-trifluoro-methanesulfo-nyloxy group, camphorsulfonyloxy group or tosyloxy group.
 13. The microarrays claimed in claim 11, wherein the photo acid group is a tosyloxy group.
 14. The microarray as claimed in claim 11, wherein the hydrophobic material is styrene, urethane, ethylene, or derivatives thereof.
 15. The microarray as claimed in claim 11, wherein the functional active copolymer comprises poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 16. The microarray as claimed in claim 11, wherein the inactive copolymer comprises poly(styrene-co-[N-(tosyloxy)-maleimide]), or poly(ethylene-co-[N-(tosyloxy)-maleimide]).
 17. The microarray as claimed in claim 11, wherein the substrate comprises a polymer polymerized by organic monomers, wherein the organic monomers are ethylene, styrene, propylene, ester, acrylic acid, acrylate, alkyl acrylic acid, or alkyl acrylate.
 18. The microarray as claimed in claim 11, wherein the substrate comprises a polymer consisting of poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 19. The microarray as claimed in claim 11, wherein the substrate comprises an inorganic material of silicon wafer, ceramic material, glass, or metal.
 20. The microarray as claimed in claim 11, wherein the substrate further comprises a photoactive polymer having a photo acid group.
 21. The microarray as claimed in claim 11, wherein the biologically active material comprises nucleic acid, oligonucleotide, peptide, peptide nucleic acid, antigen, antibody, enzyme, or protein.
 22. A method for preparing an on-spot selectively activated hydrophobic slide, comprising the steps of: preparing a hydrophobic copolymer in a solvent to obtain a solution of hydrophobic copolymer; coating the solution of hydrophobic copolymer on a substrate; removing the solvent; and photolithographically activating the hydrophobic copolymer with light to convert the hydrophobic copolymer to a functional active copolymer.
 23. The method as claimed in claim 22, further comprising applying a photo mask on the hydrophobic copolymer layer during the photolithographical activation step, wherein the mask has a pattern that covers areas not to be activated and exposes areas to be activated.
 24. The method as claimed in claim 22, wherein the hydrophobic copolymer is prepared by blending, grafting or co-polymerization of a hydrophobic material and a compound bearing a functional group protected by a protecting group, where the functional group is imide or cyclic amide, and the protecting group is a photo acid group.
 25. The method as claimed in claim 22, wherein the photo acid group is a sulfonyloxy group, N-methanesulfonyloxy group, N-trifluoro-methanesulfony-loxy group, camphorsulfonyloxy group or tosyloxy group.
 26. The method as claimed in claim 22, wherein the photo acid group is a tosyloxy group.
 27. The method as claimed in claim 22, wherein the hydrophobic material is styrene, urethane, ethylene, or derivatives thereof.
 28. The method as claimed in claim 22, wherein the hydrophobic copolymer comprises poly(styrene-co-[N-(tosyloxy)maleimide]), or poly(ethylene-co-[N-(tosyloxy)maleimide]).
 29. The method as claimed in claim 22, wherein the functional active copolymer comprises poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 30. The method as claimed in claim 22, wherein the substrate comprises a polymer polymerized by organic monomers, wherein the organic monomers are ethylene, styrene, propylene, ester, acrylic acid, acrylate, alkyl acrylic acid, or alkyl acrylate.
 31. The method as claimed in claim 22, wherein the organic substrate comprises a polymer consisting of poly(styrene-co-maleimide), or poly(ethylene-co-maleimide).
 32. The method as claimed in claim 22, wherein the substrate comprises an inorganic material of silicon wafer, ceramic material, glass, or metal.
 33. The method as claimed in claim 22, further comprising activating the substrate surface prior to the coating step.
 34. The method as claimed in claim 22, wherein the activating step comprising treating the substrate surface with an acid or a base, or treating the substrate surface by plasma activation.
 35. The method as claimed in claim 22, further comprising cleaning the substrate surface before coating the solution of hydrophobic copolymer onto a substrate.
 36. The method as claimed in claim 22, wherein the cleaning step is performed by the pretreatment with a solvent and/or sonication, wherein the solvent is surfactant, water, alcohol, or acetone.
 37. The method as claimed in claim 22, wherein the coating step comprises spin coating, screen printing, roller coating, curtain coating, or dip coating.
 38. The method as claimed in claim 22, wherein the step of removing solvent comprises vacuum evaporation, heating evaporation, or evaporation under reduced pressure. 